We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca 2ϩ -dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca 2ϩ -induced Ca 2ϩ -sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 . In the present study, we investigated whether and how cAMP affected Ca 2ϩ -dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca 2ϩ mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca 2ϩ -mediated Rho activation is dependent on class II ␣-isoform of phosphoinositide 3-kinase (PI3K-C2␣). Forskolin inhibited KCl-induced stimulation of PI3K-C2␣ activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr 850 and CPI-17 at Thr 38 in manners dependent on both PI3K and Rhokinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca 2ϩ -mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2␣, Rho, and Rho kinase in a manner independent of Ca 2ϩ and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction. Ca2ϩ plays a primary role in the regulation of smooth muscle contraction (Somlyo and Somlyo, 1994). Excitatory receptor agonist stimulation and membrane depolarization induce an increase in the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) through phospholipase C-catalyzed generation of inositol-1,4,5-trisphosphate and/or gating of plasma membrane Ca 2ϩ channels, resulting in the activation of the Ca 2ϩ -and calmodulin-dependent enzyme myosin light-chain kinase (MLCK). Receptor agonists also activate Rho and Rho kinase, which leads to inhibition of myosin phosphatase (MLCP) through mechanisms involving Rho kinase-dependent phosphorylation of the MLCP-regulatory proteins MYPT1 and CPI-17 (Noda et al., 1995;Pfitzer, 2001;Somlyo and Somlyo, 2003;Hartshorne et al., 2004). CPI-17 is phosphorylated by the protein kinase C-dependent mechanism as well (Eto et al., 1995). We and others previously dem-

The mechanism of gastrointestinal dysmotility in inflammatory bowel disease has not been clarified. In this study, we examined the mechanism involved in the inflamed distal colon isolated from a mouse model of dextran sodium sulphate-induced ulcerative colitis (DSS-treated mouse). Although substance P-induced contraction was not changed, carbachol-induced contraction was reduced in the DSS-treated mouse colon. Pre-incubation with the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) or the cyclooxygenase inhibitor indomethacin did not reverse the carbachol-induced contraction in the DSS-treated mouse colon. In semi-quantitative reverse transcription-polymerase chain reaction experiments and Western blot analysis, muscarinic M3 receptor expressions were not changed. The Ca2+ -sensitization of contractile elements induced by carbachol with GTP or GTPgammaS was reduced in the beta-escin-permeabilized DSS-treated mouse colon. Although the expression of proteins such as rhoA, ROCK1, ROCK2 or MYPT1 in smooth muscles was not changed, the expression of CPI-17, the functional protein involved in smooth muscle Ca2+ -sensitization, was significantly decreased in the DSS-treated mouse colon. These results suggest that the suppression of carbachol-induced contraction in mice with colitis is attributable at least partially to the increased activity of myosin phosphatase following the downregulation of CPI-17.

Protease‐activated receptor (PAR)‐2 plays important roles in intestinal inflammatory responses. Changes in PAR‐2‐mediated smooth muscle function may contribute pathophysiologically to the intestinal motility disorders often observed in inflammatory bowel disease (IBD). Stimulation of PAR‐2 by trypsin‐induced relaxation of carbachol‐ and KCl‐induced contractions in normal rat colonic smooth muscle was completely resolved by tissue pretreatment with apamin, but not by pretreatment with l‐NMMA or a cocktail of neuronal blockers (tetrodotoxin, hexamethonium and propranolol). In colon inflamed by dextran sodium sulphate (DSS), trypsin‐induced inhibitory effects were significantly reduced. Relaxation induced by SLIGRL‐NH2, a selective PAR‐2‐activating peptide, was also reduced in DSS‐treated rat colon. However, inhibitory effects of 1‐ethylbenzimidazolin‐2‐one, an activator of small conductance Ca2+‐activated K+ channel, were unaffected. Expression of PAR‐2 mRNA in colonic muscularis externa was significantly lower in DSS‐treated rats than in control rats. These results suggest that the PAR‐2 mediated relaxation system in colonic smooth muscle is suppressed in this experimental colitis rat model, and may contribute to motility disorders in IBD. British Journal of Pharmacology (2006) 148, 200–207. doi:

ABSTRACT. Protease-activated receptor (PAR)-2 plays important roles in intestinal inflammatory responses and also contributes to intestinal digestive motility. In the distal colon of a rat experimental colitis model, expression level of PAR-2 mRNA was decreased, and relaxation through PAR-2 activation was attenuated. This study shows the effects of proinflammatory cytokines on changes to PAR-2 in rat colonic smooth muscle using an organ culture method. Colonic inflammation was induced in rats by administering dextran sodium sulphate in drinking water. Organ culture of distal colonic smooth muscle layer of normal rat was performed for up to 3 days. In the experimental colitis rat, mRNA expression levels of proinflammatory cytokines such as IL-1 and TNF- increased with inflammation. After the incubation with IL-1 and TNF- for 3 days, trypsin (PAR-2 agonist)-induced relaxation was attenuated, simultaneous with suppression of PAR-2 mRNA expression. Conversely, in this preparation, mRNA expression levels of iNOS were significantly increased. When l-NMMA was added to the medium with IL-1 and TNF-, changes to PAR-2 by these cytokine recovered. Moreover, when samples were cultured with NOC-18 (slow-releasing NO donor) for 3 days, relaxation induced by trypsin and expression of PAR-2 mRNA were attenuated. These results suggest that suppression of PAR-2 expression under inflammatory conditions is at least partially induced by NO produced in the colonic muscularis externa by proinflammatory cytokines.KEY WORDS: colonic smooth muscle, inflammatory bowel disease, motility disorder, protease-activated receptor-2.